An impaired and defective intestinal barrier is observed in ageing (20, 22). Consistently, the electron microscope analysis shows that 100-108 weeks (wks) old ageing mice had an impaired gut integrity, as evidenced by increases in the length and/or diameter in adherens junctions, tight junctions, or desmosomes ( Figure S1A ). In addition, the expression of key genes associated with gut gap junction was significantly repressed in ageing mice ( Figure S1B ). After 1 hour of oral administration of FITC dextran to ageing mice, plasma FITC fluorescent intensity was markedly elevated, demonstrating increased intestinal permeability in ageing mice ( Figure S1C ). The abundance of IgA, which plays a critical role in preventing the leakage of microbiota (24), was significantly lower in ageing mouse gut, compared to young mice ( Figure S1D ). These results demonstrate that ageing is accompanied by the impairment of gut integrity.

Previous studies have demonstrated that microbial DNA-containing gut extracellular vesicles (mEVs) are readily translocated through an impaired gut barrier into host circulation and distal tissues (23). Consistently, the ageing-associated leaky gut resulted in the enrichment of mEVs in plasma, whereas, bacterial DNA was barely detected in the plasma EVs collected from young lean WT mice ( Figure 1A ). More importantly, as shown in Figures 1B, C and S1E , 16s rRNA was readily detected in both ageing human and mouse cardiomyocytes. To further assess whether gut mEVs are leaky from the gut lumen into host circulation and hearts in ageing mice, red fluorescent dye PKH26-labeled gut mEVs (5x10⁹ EVs/mouse) were injected into the ileum section of either young or ageing WT recipient mice. After 16 hours, PKH26 signals were readily detected in the cardiomyocytes of ageing recipient mice, thus suggesting the leakage of injected mEVs from the ageing gut ( Figure 1D ). In contrast, PKH26 fluorescence was barely detectable in young mouse hearts ( Figure 1D ).

To examine if bacterial DNA enriched in ageing heart is derived from microbiota, ageing WT mice were orally given antibiotics for 2 wks. Compared to ageing controls treated with saline, antibiotic treatment resulted in a remarkable reduction in bacterial DNA abundance in the circulation and heart ( Figures 1E, F ). We also compared the bacterial DNA species accumulated in the heart with the microbial composition of ageing WT mice. The 16s rRNA seq analysis indicates that phylum Firmicutes were the dominant microbial species in the ileum section of ageing gut ( Figures 1G , S1F–S1G , Tables S1 , S2 ). Consistently, the bacterial DNA enriched in ageing hearts was mainly derived from phylum Firmicutes ( Figure 1G ). Taken together, these data suggest that microbiota-derived mEVs are readily shuttled from the impaired intestinal barrier into the heart in the context of ageing.

Liver Vsig4⁺ Kupffer cells (KCs) are important sentinel cells that remove bacteria and their products from the bloodstream (23, 25). We observed that ageing livers contained a significantly lower amount of Kupffer cells (Clec4f⁺F4/80⁺) than young lean mouse livers, concomitant with markedly lower Vsig4 abundance on ageing KCs ( Figures 2A, B ). Previous studies have shown that Vsig4⁺ macrophages can efficiently clean gut mEVs from the bloodstream (23). After injecting PKH26-labeled gut mEVs into the tail vein of young lean Vsig4^-/- mice to mimic ageing-associated high levels of mEVs in circulation, robust red fluorescent PKH26 signals were present in the heart ( Figure 2C ). We also observed that PKH26 signals were diminished after 48 hours injection and almost gone after 72 hours ( Figure S2A ). Conversely, young lean WT mice showed few PKH26 fluorescence in cardiomyocytes after 16 hours injection of PKH26 mEVs, thus demonstrating the critical role of Vsig4⁺ cells in preventing the infiltration of mEVs into the heart ( Figure 2C ).

In addition to the liver, Vsig4⁺ macrophages also reside in other tissues such as pancreatic islets and adrenal glands (26–28). Consistently, we found that ~20% of cardiac macrophages (CD11b⁺F4/80⁺) were Vsig4 positive in young mice ( Figure S2B ), whereas, ageing hearts harbored less population of Vsig4⁺ macrophages than young mice or humans ( Figures 2D , S2C ). In addition, ageing results in a significant reduction in the cardiac macrophage population ( Figure S2D ). To further assess the ability of cardiac Vsig4⁺ macrophages to block the infiltration of gut mEVs into cardiomyocytes, we depleted Kupffer cells by injecting diphtheria toxin (DT) to young Clec4fCre⁺DTR⁺ mice (KCKO; 10-12wks old) and then intravenously injected PKH26-labeled gut mEVs into these young KCKO mice ( Figures S2E, S2F ) (29). After 24 hours, no PKH26 signal was detected in young KCKO mice treated with PKH26 EVs ( Figure S2G ). In addition, injecting gut mEVs (5x10⁸ EVs/mouse) into the jugular vein of young WT mice did not cause bacterial DNA accumulation in the hearts ( Figure 2E ). In contrast, pre-treating Vsig4 antibodies into jugular vein blocked the function of heart Vsig4⁺ macrophages of young WT mice, subsequently resulting in the infiltration of gut mEVs into cardiomyocytes ( Figure 2E ). Thus, these data suggest that cardiac macrophages exert a profound function in cleaning gut EVs.

Previous studies have demonstrated that NAD deficiency leads to ageing-associated macrophage dysfunction (14, 30). We also found that both ageing KCs and cardiac macrophages contained less abundance of key enzymes associated with the NAD salvage pathway than young mice ( Figures 2F , S2H ). To assess the effect of NAD homeostasis on Vsig4 expression, KCs isolated from ageing WT mice were treated with biosynthetic NAD precursor, NMN. After 24 hours, NMN treatment led to a significant increase in Vsig4 abundance in ageing KCs ( Figure 2G ).

Ageing is concomitant with impaired cardiac contraction. To demonstrate the regulation of microbiota on ageing cardiac functions, ageing WT mice were treated with antibiotics ( Figure S3A ). After 2 wks of treatment, we found that depletion of microbiota led to an improvement of cardiac contraction, as demonstrated by elevated levels of left ventricular systolic pressure, the derivative of pressure over time maximum, and dP/dt (the derivative of pressure over time) minimum (min) in the hearts of antibiotic-treated ageing mice ( Figures 3A–C ). In addition, the abundance of Serca2, which is critical for cardiac calcium handling for contractility, was elevated in ageing hearts after 2 wks of antibiotic treatment ( Figure 3D ).

Given that the leakage of microbiota-derived mEVs leads to microbial DNA accumulation in cardiomyocytes in ageing mice ( Figure 1 ), we next assessed the pathogenic effects of microbial DNA enrichment on cardiac functions. Young lean Vsig4^-/- mice were intravenously injected with gut mEVs (5x10⁹ EVs/mouse). After 4wks of mEV treatment, bacterial DNA levels in the hearts of young lean Vsig4^-/- mice were comparable to those of ageing WT mouse hearts ( Figure S3B ). More importantly, gut mEV treatment resulted in impaired cardiac functions, as evidenced by decreased levels of left ventricle (LV) systolic pressure, dP/dt max, and dP/dt min in the hearts of gut mEV-treated young lean Vsig4^-/- mice ( Figures 3E–G ). In addition, Serca2 expression was reduced in the heart after gut mEV treatment ( Figure 3H ). In contrast, depletion of microbial DNA cargo from gut mEVs led to non-significant changes in bacterial DNA levels in heart and cardiac functions of young lean Vsig4^-/- mice, thus demonstrating that microbial DNA is the key pathogenic cargo contributing to the effects of gut mEVs on cardiac responses ( Figures S3B , 3E–G ). In addition, after 3wks withdrawn from gut mEV treatment, heart Serca2 levels were restored ( Figure S3C ). We also found that Vsig4 knockout didn’t affect heart phenotypes in young mice ( Figures S3D–F ).

To assess the direct effects of microbial DNA on inducing cardiomyocyte dysfunction, cardiomyocytes isolated from young WT mice were in vitro treated with either gut mEVs or DNA-free EVs ( Figure S3G ). Following gut mEV-induced microbial DNA accumulation, young WT cardiomyocytes displayed a marked reduction in rates of contraction ( Figures S3H , 3I, J ). We also evaluated the state of calcium handling in cardiomyocytes after gut mEV treatment. Microbial DNA accumulation blunted the ability of young cardiomyocytes to mediate calcium homeostasis for contraction ( Figure 3K ). In contrast, depletion of microbial DNA cargos markedly reduced these effects of gut mEVs on cardiomyocyte responses ( Figures S3H , 3I, J ). Taken together, these results demonstrate that microbial DNA is a pathogenic factor inducing ageing-associated cardiac contractility abnormalities.

Previous studies have demonstrated that the infiltration of microbial DNA triggers mammalian cell inflammatory responses (23). Concomitant with a marked accumulation of microbial DNA in ageing hearts, ageing cardiomyocytes displayed greater levels of inflammation than young lean cardiac myocytes, as shown by a higher abundance of proinflammatory mediators, including Il1b, Ccl2, Ifng, and Il6, in ageing cells ( Figure 4A ). In contrast, microbiota-depleted ageing mice displayed attenuated inflammation in the heart, compared to ageing control mice ( Figure 4B ). We also found that heart abnormalities were restored in ageing WT mice after recovering microbiota from 2 wks antibiotic withdrawal ( Figures S3A , S4A, B ). To further demonstrate that microbial DNA shuttled by gut mEVs is a pathogenic factor inducing ageing-related cardiac inflammation, either young lean Vsig4^-/- or KCKO mice were intravenously injected with gut mEVs. The control young mice were treated with DNA-free EVs. After 4wks treatment, the hearts of young Vsig4^-/- mice injected with gut mEVs expressed greater levels of proinflammatory cytokines than that in control mice injected with DNA-free EVs, indicating that microbial DNA cargo within gut mEVs elevate heart inflammatory responses ( Figure 4C ). In addition, microbial DNA was barely accumulated in the cardiomyocytes of young lean KCKO mice after 4wks of intravenous treatment with gut mEVs, thus leading to comparable inflammatory responses in cardiomyocytes among young lean KCKO mice ( Figures S2F , 4D ). After the injection of gut mEVs into jugular vein of Vsig4 antibodies-pretreated young WT mice, heart inflammation levels were elevated ( Figure S4C ). We also found that in vitro treatment with gut mEVs directly promoted an inflammatory state in young WT cardiac cells, whereas, depletion of microbial DNA cargo blunted these effects of gut mEVs ( Figure 4E ).

The cGAS/STING signaling is critical to sense bacterial DNA and subsequently trigger host cellular responses (31, 32). We found that ageing cardiomyocytes expressed greater levels of cGAS and phosphorylated STING (pSTING) than young cardiac myocytes, whereas, the activation of cGAS/STING signaling in the heart was blunted after depletion of microbiota and reducing heart microbial DNA accumulation in ageing mice ( Figures 4F, G ). We also demonstrated that microbial DNA shuttled by gut mEVs plays a critical role in inducing cGAS/STING signaling activation in cardiomyocytes, as shown by elevated cGAS and pSTING in gut mEVs-treated cardiomyocytes but not in DNA-free gut EV-treated cells ( Figures S4D, E ). To further assess the importance of cGAS/STING signaling on the ability of microbial DNA to induce cardiomyocyte inflammation, young lean cGAS^-/- (cGAS knockout) cardiac cells were treated with gut mEVs in vitro. After 24 hours of treatment, the cellular inflammation state was comparable among all cGAS^-/- cells ( Figures S4F, G ). While microbial DNA was accumulated, gut mEV treatment had minimal effects on the heart inflammation of young lean cardiomyocyte-specific Sting knockout (Myh6Cre⁺Sting^f/f; Sting^ΔCM) mice pre-treated with Vsig4 antibodies ( Figures 4H , S4H, I ). Taken together, these data suggest that microbial DNA induces ageing-related cardiac inflammation through the activation of cGAS/STING signaling.

Previous studies have demonstrated that inflammation is an important factor initiating heart dysfunctions (33, 34). While microbial DNA enrichment occurred in the cardiomyocytes of ageing cGAS^-/- mice, these cGAS^-/- ageing cardiac cells displayed lower levels of inflammation but better contractile activity than ageing WT cardiomyocytes ( Figures S5A , 5A–C ). In addition, ageing cGAS^-/- cardiomyocytes contained less pSTING than ageing WT cells ( Figure S5B ). Knockout of cGAS also blunted the effects of ageing on cardiac calcium handling, as evidenced by greater Serca2 abundance in ageing cGAS^-/- cardiomyocytes ( Figure 5D ). We next assessed the importance of cGAS/STING-induced inflammation on the ability of microbial DNA to induce ageing-related cardiac dysfunctions. In young lean Sting^ΔCM mice, concomitant with their blunted inflammatory responses to microbial DNA accumulation, gut mEV treatment had minimal effects on Serca2 abundance and contractility in the heart ( Figures S4F , 5E–G ). Thus, these data suggest that cGAS/STING-mediated inflammation plays a critical role in microbial DNA-induced ageing cardiac dysfunctions.

Given that Vsig4⁺ macrophages efficiently clean bacterial products from the bloodstream, we next evaluated the effects of Vsig4 overexpression on ageing-associated heart microbial DNA accumulation and cardiac functions. To overexpress Vsig4, ageing WT mice were intravenously injected with lentivirus carrying the VPR system (including three transcriptional activators VP64, p65, and Rta) linked to the C-terminal end of deactivated Cas9 (dCas9) and gRNA-Vsig4 TSS (Transcription start site; Vsig4oe). After 2 wks of injection with these lentiviruses, Vsig4 abundance was elevated in the livers and hearts of ageing mice ( Figures S6A, B ). Following the recovery of the Vsig4⁺ macrophage population, we observed that the bacterial DNA abundance was significantly reduced in the hearts of ageing mice, concomitant with lower levels of cGAS/STING activation and proinflammatory cytokines in the heart ( Figures 6A–C ). In addition, ageing mice displayed improved cardiac contraction and Serca2 abundance in the heart after restoring Vsig4⁺ macrophages ( Figures 6D–G ). Therefore, these data demonstrate that restoration of the Vsig4⁺ macrophage population is an efficient way to blunt microbial DNA-induced heart dysfunction in ageing mice.

cGAS^-/- (Stock No. 026554) Sting-flox (Stock No. 031670), iDTR (Stock No. 007900; Cre-inducible expression of diphtheria toxin receptor), Myh6-Cre (Stock No. 011038), Clec4f-Cre (Stock No. 033296) mice were received from the Jackson Laboratory. Vsig4^-/- mice (C57BL/6J-Vsig4^em1Smoc) were received from Shanghai model organisms. Vsig4 wild-type (WT) mice were produced by crossing Vsig4 heterozygous mice together. Ageing mice used in these studies were 100-108 wks old, and young mice were 8-12 wks old. To block Vsig4 function, young Myh6Cre⁺Sting^f/f male mice were injected with purified Vsig4 antibodies before gut mEV treatment. through tail vein injection. All mice were maintained on a 12/12 hr light-dark cycle. All animal procedures were performed in accordance with the University of California, San Diego Research Guidelines for the Care and Use of Laboratory Animals, and all animals were randomly assigned to cohorts when used.

The intestinal EVs were prepared from small intestine lumen contents of ageing WT mice with sterile tools. Debris and dead cells in the lumen contents were removed by centrifugation at 1,000 x g for 10 min and then passed through a 0.2 µm filter. Then, the supernatant was added with a mixture of antibiotics (0.5 mg/mL vancomycin HCl, 1 mg/mL ampicillin sodium salt, 1 mg/mL metronidazole, 1 mg/mL neomycin sulfate, and 1mg/mL gentamycin sulfate; Sigma) and then ultracentrifuged at 100,000 x g for 4 hours at 4°C with a Type 70 Ti fixed-angle rotor (Beckman Coulter). The EV-containing pellet was resuspended in 1 mL of sterile PBS and passed through a 0.2 µm filter to remove large particles in a sterile hood. The particle size and concentration of intestinal EVs were detected by NanoSight analysis (Malvern Instruments).

PKH26 fluorescent dye using the PKH26 fluorescent cell linker kit (Cat. No. PKH26GL-1KT, Sigma). After PKH26 staining, the EVs were washed with sterile PBS and collected by ultracentrifugation (100,000 x g for 2 hours) at 4°C. Finally, PKH26 labeled EVs were resuspended in sterile PBS and passed through a 0.2 µm filter.

PKH26-labeled EVs (5x10⁹ EVs per mouse) were delivered to either young or ageing WT recipient mice through injection into the ileum section. After anesthetized, the ileum section was exposed through making a small incision at the abdominal area, and then 150 µL of saline containing EVs were injected into ileum. Following closure of incisions, sites will be treated with 10% povidone-iodine solution. After 16 hours of injection, hearts were collected to detect PKH26 red fluorescent signals.

Mice will be anesthetized with ketamine (25 mg/kg), acepromazine (1 mg/kg) and xylazine (2 mg/kg) via intramuscular injection. Incision sites will be shaved and cleaned with isopropyl alcohol and a 10% povidone-iodine solution. The right jugular vein will be cleared of surrounding tissue and a sterilized dual microreathane catheter (Type MRE-025) filled with heparinized saline (100 U/mL) will be inserted ~1 cm into vessel and secured with double silk ligatures. Catheter ends will then be tunneled subcutaneously to the mid-scapular region, externalized, placed in a protective silastic tubing (0.18 cm), and sutured to the skin. Following closure of incisions, sites will be treated with 10% povidone-iodine solution and animals will be given 1cc 0.9% sterile saline subcutaneously for rehydration. Lidocaine (2.5%) will be administered twice a day for 5 days post-surgery. To block the function of Vsig4+ macrophage in hearts, Vsig4 antibodies (0.5 mg/ml) were injected into heart through the jugular vein catheter. Control mice were injected with IgG antibodies. After 24 hours, PKH26-labeled gut mEVs (5x10⁸ EVs/mouse) were injected into the jugular vein catheter of these antibodies-treated mice.

Mice were fasted for 6 hours and then orally gavaged with FITC dextran (600mg/Kg body weight; Sigma) from a 125mg/mL solution (48, 49). Plasma samples were collected after 1 hour FITC dextran treatment and used to measure the intensity of FITC by fluorescence spectroscopy (excitation at 485nm and emission at 535nm) relative to a linear standard curve made using diluted FITC dextran solution in plasma from untreated mice.

The isolated gut EVs from ageing WT mice were resuspended in sterile PBS. Then, as previously described (23, 50–52), these EVs were loaded into a Gene Pulser/micropulser Cuvettes (Bio-Rad) for electroporation (GenePulser Xcell electroporator, Bio-Rad) and treated with DNase I (300U) for 30 mins, 37°C.

Calcium-tolerant adult cardiomyocytes were isolated from ventricular tissue of mice by standard enzymatic digestion (53, 54). Briefly, isolated hearts were perfused using a Ca²⁺-free Tyrode solution containing (in mM) 126 NaCl, 4.4 KCl, 1.2 MgCl₂, 0.12 NaH₂PO₄, 4.0 NaHCO₃, 10 HEPES, 30 2,3-butanedione monoxime, 5.5 glucose, 1.8 pyruvate, and 5.0 taurine (pH 7.3) for 5 min, followed by 0.9–1.0 mg/mL collagenase (type II, Worthington) for 20 min. Hearts were transferred to tubes containing fresh collagenase for an additional 10 min in a 37°C water bath. The heart tissue was mechanically dispersed and rinsed with gradually increasing extracellular calcium to 1 mM. The cells were plated on 24 × 50-mm, No. 1 glass coverslips coated with laminin.

After anesthetized and endotracheal intubation, mice were connected to a volume-cycled rodent ventilator. The anterior neck and abdomen were shaved. A subcostal incision was performed in the abdomen. The diaphragm was incised by a transverse substernal approach leaving the pericardium intact. The left ventricle was entered through an apical stab with a 25 1/2 G needle, followed immediately by a 1F Millar conductance-micromanometer (Millar Instruments). The catheter was positioned in the left ventricle and monitored to verify correct placement. Pressure recordings were obtained and analyzed to obtain left ventricular pressure (LVP), rate of contraction (dP/dt max) and rate of relaxation (dP/dt min).

Cardiac myocyte (CM) contractility is measured with a state-of-the-art integrated myocyte contractility workstation (IonOptix LLC). This method combines brightfield imaging with a high-speed camera recording system, along with sarcomere and cell-length detection algorithms, and application-specific analysis software to output reliable quantification of cardiomyocyte contractile function at the sarcomere level. Analysis outputs include determination of ±ΔL/Δt (dL/dt), representing peak shortening/relengthening velocities, and percent shortening for both sarcomere and cell length. CM contractility was measured after 36 hours of treatment with gut EVs.

KCs (Clec4f⁺F4/80⁺) isolated from ageing WT mice were seeded in a 24-well plate (0.5x10⁵/well) and cultured with nicotinamide mononucleotide (NMN; 1mM). After 24 hours, cells were used for qPCR assays.

Bacterial DNA was isolated from ileum lumen content and heart samples using the ZymoBIOMICS DNA extraction kits. Bacterial 16s ribosomal RNA gene targeted sequencing was performed using the Quick-16S™ NGS Library Pre Kit (Zymo Research). The bacterial 16s primers amplified the V3-V4 region of the 16s rRNA gene. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned with the Select-a-Size DNA Clean & Concentrator™ (Zymo Research), then quantified with TapeStation^® (Agilent Technologies) and Qubit^® (Thermo Fisher). The final library was sequenced on an Illumina MiSeq with a v3 reagent kit (600 cycles). The sequencing was performed with 10% PhiX spike-in. For bioinformatics analysis, unique amplicon sequences variants were inferred from raw reads using the DADA2 pipeline (55). Potential sequencing errors and chimeric sequences were also removed with the DADA2 pipeline. Chimeric sequences were also removed with the DADA2 pipeline. Taxonomy assignment was performed using Uclust from Qiime v.1.9.1 with the Zymo Research Database, a 16s database that is internally designed and curated. Composition visualization, alpha-diversity, and beta-diversity analysis were performed with Qiime v.1.9.1 (56). If applicable, a taxonomy that has significant abundance among different groups was identified by LEfSe using default settings (57). Sequencing raw data supporting these studies can be found in the Sequence Read Archive database under accession number PRJNA859808.

To deplete Kupffer cells, diphtheria toxin (DT) was intraperitoneally (i.p.) injected into Clec4fCre⁺DTR⁺ lean mice (crossbreeding Clec4fCre mice and iDTR mice) daily for three days (200 ng/mouse). Thereafter, these mice were injected with DT (200 ng/mouse) every two days to prevent KC recovery (26).

Plasmids containing deactivated Cas9-VPR (VP64, p65, and Rta) system (Cat. No. CAS11915) or guide RNA for Vsig4 TSS (Cat. No. GSGM11893-247477006) were obtained from Horizon Discovery (58–60). The lentivirus packing these plasmids were prepared by the UCSD Vector core. 100wks old ageing WT mice were intravenously injected with these lentiviruses (1x10⁸ particles/mouse). Control mice were treated with lentivirus containing dCas9-VPR only. After 2 wks, Vsig4 expression in livers and hearts was evaluated by Western blot analysis.

Ageing WT mice (100-108wks old) were subject to oral gavage (twice per week; 200 µL per mouse) with a mixture of broad-spectrum antibiotics (0.5 mg/mL vancomycin HCl, 1 mg/mL ampicillin sodium salt, 1 mg/mL metronidazole, 1 mg/mL neomycin sulfate, and 1mg/mL gentamycin sulfate; Sigma) (48). Control mice were treated with water alone. The ageing WT mice in the antibiotic withdrawal group were fed ad libitum without antibiotic treatment for another 2 wks.

Bacterial DNA was assessed by qPCR using a Femto Bacterial DNA Quantification Kit by following the manufacturer’s instructions. Briefly, Bacterial DNAs were extracted from cells, tissues, or plasma samples using the ZymoBIOMICS DNA extraction kits according to the manufacturer’s instructions. The concentration of bacterial DNA in each sample was calculated from the standard curve.

Mouse hearts were snap frozen in optimum cutting temperature (O.C.T., Fisher Healthcare) with dry ice. Tissue cryo-sections were prepared and fixed with pre-cold acetone for 20 min. Slides were blocked with 5% normal donkey serum for 60 min at RT. Then, the samples were incubated with antibodies diluted 1:100 in PBS at 4°C overnight. After washing, nuclei were stained with DAPI (4’,6-Diamidino-2-28 phenylindole dihydrochloride) for 10min at room temperature. Mounting media and coverslips were then added to slides for imaging. Images were acquired on a Keyence Fluorescent Microscope and were processed with ImageJ (NIH, Bethesda, MD).

We performed RNAscope ISH to detect 16s rRNA. Mouse hearts were frozen in O.C.T with dry ice. Human heart sample information is presented in Table S3 . Tissue sections were fixed with 4% PFA for 15 min at 4°C and then dehydrated with 50%, 70%, and 100% ethyl alcohol gradients for 5 min each at room temperature. Next, tissue sections were treated with hydrogen peroxide and protease IV at room temperature for 10 min each. 16s RNA probes (Cat. No. 464461, Advanced Cell Diagnostics) were then added for 2 hours at 40°C. Signal amplification and detection reagents were applied sequentially and incubated in AMP 1, AMP 2, AMP 3, HRP-C1 (RNAscope® Multiplex fluorescent reagent kit v2, Cat. No. 323100, Advanced Cell Diagnostics), Opal 520 (Cat. No. PNFP1487001KT, Akoya Biosciences), or Opal 690 (Cat. No. FP1497001KT, Akoya Biosciences). Then, slides were immediately processed for immunofluorescence, and images were taken with a Leica SP8 Confocal microscope.

The liver and heart were perfused to get single cells which were then stained with fluorescence-tagged antibodies. KCs were CD45⁺Clec4f⁺F4/80⁺ cells, and cardiac macrophages were CD45⁺CD11b⁺F4/80⁺ cells. These cells were analyzed by an MA900 flow cytometer (SONY). Data were analyzed using Flowjo software. Clec4f (Cat. No. 156804), F4/80 (Cat. No. 123114), CD11b (Cat. No. 101235), and CD45 (Cat. No. 103116) antibodies were received from Biolegend, and Vsig4 antibody (Cat. No. 17-5752-82) was from ThermoFisher Scientific. The concentration of antibodies (0.25µg per 10⁶ cells) for sample staining were used.

Total RNA was extracted using the RNA extraction protocol according to the manufacturer’s instructions. cDNA was synthesized using MultiScribe Reverse Transcriptase and random primers (High-capacity cDNA reverse transcription kit, Cat. No. 4368813, ThermoFisher Scientific). qPCR was carried out in 10 μl reactions using iTaq SYBR Green supermix (Cat. No. 172-5125, Bio-Rad) on a StepOnePlus Real-Time PCR Systems (ThermoFisher Scientific). The data presented correspond to the mean of 2-ΔΔCt from at least three independent experiments after being normalized to β-actin.

Cells or tissues were homogenized in 1x RIPA buffer supplemented with protease and phosphatase inhibitors. Equal amounts of cell lysate proteins (30 μg protein per lane for detection) from each biological replicate were subjected to western blotting. Using the ChemiDoc XRS imaging system (BioRad), the protein bands on blots were detected with the SuperSignal West Pico Chemiluminescent Substrate (Cat. No. 34077, ThermoFisher Scientific). Protein bands were analyzed using Image Lab software (BioRad). We normalized phosphorylated protein to total protein bands or normalized protein expression to housekeeping protein bands. Western blot data in figures and supplemental figures are all representatives of more than three independent experiments. pSTING (Cat. No. 72971), STING (Cat. No. 50494), cGAS (Cat. No. 316595), and Serca2 (Cat. No. 4388S) antibodies were bought from Cell Signaling Technology. Vsig4 antibody (Cat. No. 17-5752-82) was obtained from ThermoFisher Scientific. All primary antibodies were diluted at 1:1000.

Production of lentivirus vectors: HIV1-based lentivirus vectors were produced by transient co-transfection of 293T cells maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FCS. 293T cells in ten 150 mm dishes were co-transfected by the polyethyleneimine method with gRNA vector plasmid, pLP1 (gal-pol) and pLP2 (Rev) (Invitrogen), and pCMV-G (61). Conditioned media on day 1, 2, and 3 post-transfection were collected, filtered through a 0.45 µm filter, and concentrated by centrifugation at 7000 rpm for 16 hours at 4°C with Sorvall GS-3 rotor. The resulting pellets were resuspended with buffer containing 10 mM Tris HCl, pH 7.8, 1 mM MgCl_2, and 3% sucrose. Titering of HIV1 vectors by real-time Q-PCR: HIV1-CMV-GFP vector (1x10⁹ iu/mL) was used as the standard. HEK293 cells in a 6-well plate were infected with different amounts of viruses in the presence of polybrene (4 µg/mL). Infected cells were passaged once every 4 days and cell DNAs were prepared on day 14 post-infection by the DNeasy Blood & Tissue kit (Qiagen Science, MD). Real-time Q-PCR was performed using a primer set selected from the WPRE sequence.

To assess whether the means of two groups are statistically different from each other, an unpaired two-tailed Student’s t-test was used for statistical analyses using Prism8 software (GraphPad software v8.0; Prism, La Jolla, CA). P values of 0.05 or less would be considered to be a significant difference. Degrees of significance was indicated in each of the figure legends.